Means and methods for manufacturing highly pure neurotoxin

ABSTRACT

The present invention relates to an antibody which specifically binds to unprocessed and/or partially processed neurotoxin polypeptide or an antibody which specifically binds an epitope consisting of a peptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 16 and to methods for the manufacture of such antibodies. Moreover, the present invention relates to a composition comprising processed neurotoxin polypeptide free of unprocessed or partially processed neurotoxin polypeptide and a method for manufacturing said neurotoxin polypeptide based on the antibodies of the invention. The present invention also relates to the use of the aforementioned antibody for separating processed neurotoxin polypeptides from unprocessed or partially processed neurotoxin polypeptides or for determining unprocessed or partially processed neurotoxin polypeptides. The present invention relates to a method for the manufacture of a medicament.

The present invention relates to an antibody which specifically binds tounprocessed and/or partially processed neurotoxin polypeptide or anantibody which specifically binds an epitope consisting of a peptidehaving an amino acid sequence as shown in any one of SEQ ID NOs: 1 to16. Moreover, the present invention relates to a method formanufacturing a Neurotoxin polypeptide, comprising the steps of,contacting a solution containing a mixture of proteolytically processed,partially processed and/or unprocessed neurotoxin polypeptides with anagent that specifically binds to unprocessed or partially processedNeurotoxin polypeptides but not to the processed neurotoxin polypeptidesunder conditions which allow binding of said agent to the unprocessed orpartially processed neurotoxin polypeptides whereby an antigen-agentcomplex is formed, and removing the formed antigen-agent complex,whereby a solution containing processed neurotoxin polypeptide free ofunprocessed or partially processed neurotoxin polypeptide is obtained.The present invention also relates to the use of the aforementionedantibody for separating proteolytically processed neurotoxinpolypeptides from unprocessed or partially processed neurotoxinpolypeptides. The present invention relates to a method for themanufacture of a medicament comprising the steps of the above method andthe further step of formulating the proteolytically processed neurotoxinpolypeptides as medicament. Furthermore, the present invention relatesto a composition comprising the proteolytically processed neurotoxinpolypeptide obtainable by the aforementioned method.

Clostridium botulinum and Clostridium tetani produce highly potentneurotoxins, i.e. botulinum toxins (BoNTs) and tetanus toxin (TeNT),respectively. These Clostridial neurotoxins (CNTs) specifically bind toneuronal cells and disrupt neurotransmitter release. Each toxin issynthesized as an inactive unprocessed approximately 150 kDasingle-chain protein. The posttranslational processing involvesformation of disulfide bridges, and limited proteolysis (nicking) bybacterial protease(s). Active dichain neurotoxin consists of two chains,an N-terminal light chain of approx. 50 kDa and a heavy chain of approx.100 kDa linked by a disulfide bond. CNTs structurally consist of threedomains, i.e. the catalytic light chain, the heavy chain encompassingthe translocation domain (N-terminal half) and the receptor bindingdomain (C-terminal half), see Krieglstein 1990, Eur J Biochem 188, 39;Krieglstein 1991, Eur J Biochem 202, 41; Krieglstein 1994, J ProteinChem 13, 49.

Clostridium botulinum secretes seven antigenically distinct serotypesdesignated A to G of the botulinum neurotoxin (BoNT). All serotypestogether with the related tetanus neurotoxin (TeNT) secreted byClostridium tetani, are Zn²⁺-endoproteases that block synapticexocytosis by cleaving SNARE proteins. CNTs cause the flaccid muscularparalysis seen in botulism and tetanus, see Fischer 2007, PNAS 104,10447.

Despite its toxic effects, botulinum toxin complex has been used as atherapeutic agent in a large number of diseases. Botulinum toxinserotype A was approved for human use in the United States in 1989 forthe treatment of strabism, blepharospasm, and other disorders. It iscommercially available as a Botulinum toxin A protein preparation, forexample, under the tradename BOTOX (Allergan Inc) under the tradenameDYSPORT (Ipsen Ltd). For therapeutic application the complex is injecteddirectly into the muscle to be treated. At physiological pH, the toxinis released from the protein complex and the desired pharmacologicaleffect takes place. An improved BoNT/A preparation being free ofcomplexing proteins is available under the tradename XEOMIN (MerzPharmaceuticals GmbH). The effect of Botulinum toxin is only temporary,which is the reason why repeated administration of Botulinum toxin maybe required to maintain a therapeutic affect.

The Clostridial neurotoxins weaken voluntary muscle strength and areeffective therapy of strabism, focal dystonia, including cervicaldystonia, and benign essential blepharospasm. They have been furthershown to relief hemifacial spasm, and focal spasticity, and moreover, tobe effective in a wide range of other indications, such asgastrointestinal disorders, hyperhidrosis, and cosmetic wrinklecorrection, see Jost 2007, Drugs 67, 669.

For the manufacture of Clostridial neurotoxins, the purification of theneurotoxin containing fermentation solution is of particular importance.In this context, different precipitation- and extraction steps followedby a concentration step and further distinct chromatographic steps areusually applied in order to obtain purified neurotoxin, see DasGupta1984, Toxicon 22, 415; Sathyamoorthy 1985, J Biol Chemistry 260, 10461.Currently, available neurotoxin preparations comprise, in addition tothe desired active (processed) neurotoxin, a proteolytically unprocessedprecursor and/or partially processed neurotoxin polypeptide. Theproteolytically unprocessed precursor or partially processed polypeptidediffers from the active (processed) neurotoxin polypeptide in a sequenceof only a few amino acids. Therefore, they can hardly be distinguishedbased on their chemical and physical properties. On the other hand, theratio of proteolytically unprocessed precursor and/or partiallyprocessed neurotoxin polypeptide of the total protein ratio is stillsignificant in such preparations. Said ratio is due to the biologicalsystem, and is determined by the biosynthesis and the conditions of thefermentation process. Thus, the amount of undesired proteolyticallyunprocessed precursor and/or partially processed Neurotoxin polypeptidein Neurotoxin preparations is predefined and, currently, ratherdifficult to reduce.

Means and methods for reducing the amount of the unprocessed and/orpartially processed neurotoxin polypeptides and thereby improving thequality of neurotoxin preparations are highly desirable but not yetavailable.

Thus, the technical problem underlying the present invention may be seenas the provision of means and methods for improving the manufacture ofneurotoxin polypeptides by complying with the aforementioned needs. Thetechnical problem is solved by the embodiments characterized in theclaims and herein below.

The present invention relates to an antibody that specifically binds anepitope consisting of a peptide having an amino acid sequence as shownin any one of SEQ ID NOs: 1 to 16.

The term “antibody” as used herein encompasses a monoclonal antibody, apolyclonal antibody, a single chain antibody, a human, humanized,primatized, or chimerized antibody, a bispecific antibody, a syntheticantibody, chemically or enzymatically modified derivatives, a fragmentof any of said antibodies or aptamers consisting of naturally occurringand/or chemically modified nucleic acids. Fragments of said antibodiesinclude F(ab′)₂, F(ab), Fv or scFv fragments or chemically orenzymatically modified derivatives of any of these fragments. Theantibody of the present invention shall specifically bind to the epitopeconsisting of the aforementioned peptide if the said peptide iscomprised by the partially processed or the unprocessed neurotoxinpolypeptide.

The term “epitope” as in accordance with the present invention relatesto the antigenic determinant which is recognized by the antibody of thepresent invention. It consists of a peptide having an amino acidsequence as shown in any one of SEQ ID NOs: 1 to 16. The aforementionedepitopes represent, in an aspect of the invention, peptides which areflanked by the cleavage sites for neurotoxin processing enzymes or whichcover the cleavage site(s), see tables 1 and 2 below. The epitope is, inan aspect of the invention, comprised by a proteolytically unprocessedneurotoxin polypeptide or by a partially processed neurotoxinpolypeptide. The partially processed neurotoxin polypeptide may eitherbe the light chain of the neurotoxin polypeptide elongated with thepeptide sequences as shown in any one of SEQ ID NOs: 1 to 8, or theheavy chain of the neurotoxin polypeptide elongated with the peptidesequences as shown in any one of SEQ ID NOs: 1 to 8. Due to the presenceof said epitope, the unprocessed or partially processed neurotoxinpolypeptides can be specifically bound by the antibody.

TABLE 1 Amino acid sequences of the epitopes and of thefull length polypeptides of the Neurotoxin serotypes SequenceNeurotoxin/ SEQ ID NO: of the Cleavage Bacterial (full length Accession-SEQ ID NO: excised peptide sites strain Neurotoxin) NO: 1^(b) TKSLDKGYNKK438/T439 BoNT/A 17 ABD65472 K448/A449 (Hall/62A) 2^(c) CKSVKAPGICK441/A442 BoNT/B 18 BAE48264 (Okra) 3^(d) SLYNK R444/S445 BoNT/C1 19BAA89713 K449/T450 (C-6814) 4^(d) NSR K442/N443 BoNT/D 20 BAA90661R445D446 (CB16) 5^(e) GIR K419/G420 BoNT/E 21 CAA43999 R422/K423(Beluga) 6^(d) KGTK R435/K436 BoNT/F 22 CAA73972 K439/A440 (NCTC10281)7  NGTK nn BoNT/G 23 CAA52275 8^(a) ENLYNR R449 TeNT 24 P04958(z. T. R445) ^(a)Krieglstein et al. 1991, Eur J Biochem 202, 41-51.;Krieglstein et al. 1990, Eur J Biochem 188, 39-45. ^(b)Beecher andDasGupta 1997, J Protein Chem 16, 701-712.; Krieglstein et al. 1994, JProtein Chem 13, 49-57. ^(c)Antharavally and DasGupta 1998, J ProteinChem 17, 417-428. ^(d)Sagane et al. 1999, J Protein Chem 18, 885-892.^(e)Antharavally and DasGupta 1997, J Protein Chem 16, 787-799.

TABLE 2 Amino acid sequences including the cleavagesites of the Neurotoxin serotypes SEQ Neurotoxin IDSequence including cleavage sites (Bacterial NO: (highlighted) Strain) 9KLLCVRGIITSKTKSLDKGYNKALN....DLCIKV BoNT/A (Hall/62A) 10IQMCKSVKAPG...................ICIDV BoNT/B (Okra) 11TKFCHKAIDGRSL....YNKTL......DCRELLV BoNT/C1 (C-6814) 12TKVCLRLTK.........NSRD......SDTCIKV BoNT/D 13IRFCKNIVSVKG......IRK........SICIEI BoNT/E (Beluga) 14VKFCKSVIPRKG......TKAP......PRLCIRV BoNT/F (NCTC10281) 15IAMCKPVMYKNT......GKS........EQCIIV BoNT/G 16IGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKI TeNT

The term “specifically binds” means that the antibody of the presentinvention does not cross react to a significant extent with otherepitopes either on said partially processed, or on said unprocessedneurotoxin polypeptides, or on other polypeptides in general. In anaspect of the invention, the antibody of the present invention does notcross react with said active, completely processed neurotoxinpolypeptide. Epitope specificity is an important characteristic of theantibody of the present invention. Specificity of the antibody withrespect to the partially processed or unprocessed neurotoxin versus theprocessed neurotoxin shall be, in an aspect, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%. Specific binding can be testedby various well known techniques including, e.g., competition studies.Another important characteristic is the sensitivity of the antibody.Sensitivity shall be, in one aspect of the invention, such that at least70%, at least 80%, at least 90%, at least 95% of the processedneurotoxin comprised by a sample is bound. Sensitivity can be tested bywell known techniques. Those skilled in the art will be able todetermine operative and optimal assay conditions for each determinationby employing routine experimentation. Conventional techniques forbinding studies include radioimmunoassay, ELISA, equilibrium dialysis,isothermal microcalorimetry, BIACORE® assays (surface plasmonreasonance, SPR) or other surface adsorption methods. The BIACORE® SPRsystem measures the antibody-antigen interaction. SPR response reflectsa change in mass concentration at the detector surface as analytes bindor dissociate. Based on SPR, real-time BIACORE® measurements monitorinteractions directly as they occur, see BIAapplications Handbook,version AB (reprinted 1998), BIACORE® code No: BR-1001-86; BIAtechnologyHandbook, version AB (reprinted 1998), BIACORE® code No: BR-1001-84. Thebinding properties such as sensitivity of an antibody of the presentinvention may, in principle, be determined by binding studies using animmobilized antigen (the ligand) presented on a sensor surface. Theantibody to be tested (the analyte) will be provided in the mobilephase, i.e. in a solution. In some cases, the antigen is attachedindirectly to the surface through binding to another immobilizedmolecule which is referred as the capturing molecule. When the antibodyis injected in a discrete pulse across the surface with the immobilizedantigens, essentially three phases can be subdivided: (i) Association ofantibody with the antigen during sample injection; (ii) Equilibrium orsteady state during sample injection, where the rate of antibody bindingis balanced by dissociation from the antibody-antigen complex; (iii)Dissociation of antibody from the surface during buffer flow. It will beunderstood that such an assay can alternatively performed withimmobilized antibodies to be investigated and an antigen containingsolution as the mobile phase. The association and dissociation phasesprovide information on the kinetics of analyte-ligand interaction (k_(a)and k_(d), the rates of complex formation and dissociation,k_(d)/k_(a)=K_(D)). The equilibrium phase provides information on theaffinity of the analyte-ligand interaction (K_(D)). In an aspect of theinvention, the antibody of the present invention has a KD of less than0.5 μM, in an aspect, less than 0.05 μM and, in another aspect, lessthan 0.02 μM.

The antibody as referred to in the present invention can be manufacturedby using methods which are described, e.g., in Harlow and Lane“Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988.Monoclonal antibodies can be prepared by the techniques originallydescribed in Köhler 1975, Nature 256, 495, and Galfré 1981, Meth Enzymol73, 3. Said techniques comprise the fusion of mouse myeloma cells tospleen cells derived from immunized mammals. Antibodies can be furtherimproved by techniques well known in the art. For example, surfaceplasmon resonance as employed in the BIACORE® system can be used toincrease the efficiency of phage antibodies which bind to theaforementioned epitope within proteolytically unprocessed neurotoxinpolypeptide, see Schier 1996, Human Antibodies Hybridomas 7, 97;Malmborg 1995, J. Immunol. Methods 183, 7.

In an aspect of the invention, the antibody according to the antibody ofthe present invention is, in one aspect, produced by using anoligopeptide comprising the aforementioned epitope. Such an oligopeptidecan be produced synthetically or by recombinant expression.Alternatively, the antibody of the invention can be produced by applyingnatural occurring unprocessed or partially processed neurotoxinpolypeptide. In the latter case, it is to be understood that theresulting antibodies shall be further tested for specificity withrespect to the unprocessed and/or partially processed neurotoxinpolypeptide(s). In a further aspect of the invention, a monoclonalantibody of the invention is produced by using partially processed orunprocessed neurotoxin polypeptide which can be treated by a detergentin order to make the epitope immunologically available. However, it willbe understood that in a case were the antibody shall be directed againsta conformational epitope, no such detergent treatment shall be carriedout. In a further aspect, immune-stimulation agents such as keyholelimpet hemocyanin (KLH) may be also applied in such process, especiallywhen using a synthetic oligopeptide.

The antibody as referred to in the present invention can be used, forexample, for affinity chromatography, immunoprecipitation, andimmunolocalization of the partially processed and/or unprocessedneurotoxin polypeptide as well as for the monitoring of the presence ofsaid polypeptide in samples or in recombinant organisms.

In an aspect of the invention, the partially processed and/orunprocessed neurotoxin polypeptide is from Clostridium spp. In anotheraspect of the invention, it is from Clostridium botulinum selected fromthe group of Clostridium botulinum ATCC 3502, Clostridium botulinum ATCC3502—Hall strain. The primary structure of the said unprocessedneurotoxin polypeptide from Clostridium botulinum is disclosed inKrieglstein 1994, J Protein Chem 13, 49.

Clostridium spp. as referred to herein is the genus of Gram-positive,endospore-forming, obligate anaerobic bacteria which belong to theFirmicutes. Clostridial neurotoxins may be produced by phenotypic andgenetic different clostridia belonging to the species Clostridiumbotulinum, Clostridium butyricum, Clostridium barati, and Clostridiumtetani. Clostridium botulinum as used herein is specie of a rod shaped,Gram-positive, obligate anaerobic bacterium which produces, besides theneurotoxins, oval, subterminal endospores, and is commonly found insoil.

Moreover, in a further aspect of the antibody of the present invention,said antibody is bound to a polypeptide carrier. In an aspect of theantibody of the present invention, the said polypeptide carrier isselected from the group consisting of: a FC-binding protein, Protein Aand Protein G and an antibody which specifically binds to the antibodyof the present invention. This may be for example, in an aspect, anantibody which is species specific. Such antibody specifically binds tothe FC portion or F(ab) of the antibody of the invention. In anotheraspect of the antibody of the present invention said polypeptide carrieris Protein A from Staphylococcus aureus. The said polypeptide carriercan be used, in an aspect of the invention, for isolating the antibodyof the present invention.

Moreover, in a further aspect of the antibody of the present invention,said antibody is bound to a matrix. In an aspect, said matrix is a solidmatrix.

The term “bound” as used herein, relates to any type of connectionbetween the antibody and the matrix as long as the said connection doesnot interfere essentially with binding of the antibody to the partiallyprocessed and/or unprocessed neurotoxin polypeptide. Said connection maybe made by interactions including indirect or direct, non-reversible orreversible, physical and chemical, electrostatic, and/or covalent bonds.In an aspect, the antibody is covalently linked, either directly or viaa linker molecule, to the matrix.

The term “matrix” as used in accordance with the present inventionrefers to a three dimensional structure or spatial arrangement capableof binding an antigen or an antibody. Well-known matrices comprisepolypeptides, glass, polystyrene, polypropylene, polyethylene,polyethylene glycol (PEG), dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, gabbros, and magnetite. A solidmatrix is, in an aspect of the invention, a polysaccharide matrixselected from the group consisting of: sepharose, sephadex; agarose,sephacell, micro-cellulose, and alginate-beads. In another aspect, saidsolid matrix can consist of glass-beads, and/or polypeptide matrices.

The antibody may be bound to the said matrix via a linker, includingsmall molecule compounds, peptide linker molecules and beads. The matrixcan have virtually any possible structural configuration or arrangementas long as the coupled antibody is capable of binding to its antigen.Thus, the matrix may be spherical, as in a bead, or cylindrical, as inthe inside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be irregular or flat such as a sheet,test strip, etc. In one aspect the said supports include polystyrenebeads.

The aforementioned matrix, in an aspect of the invention, has at leastone binding site for the antibody of the present invention. In a furtheraspect of the invention, said matrix has additional binding sites forfurther antibodies which recognize other epitopes. In an aspect, saidepitopes are other epitopes which allow for specific binding of thepartially processed and/or unprocessed neurotoxin polypeptide. Furtherantibodies immobilized on the matrix also encompass antibodies whichrecognize bacterial polypeptides other than the neurotoxin polypeptides.Such further antibodies comprised by the matrix may be used to removefurther undesired polypeptides and, thus, for further purificationpurposes of a Neurotoxin preparation. However, it is to be understoodthat in a further aspect the processed neurotoxin shall not bespecifically bound by the antibodies immobilized on the matrix.

The aforementioned antibody of the present invention is suitable for themanufacture of processed neurotoxin polypeptide because it specificallybinds to the above characterized epitope thus enabling the binding ofthe partially processed or the unprocessed neurotoxin polypeptide andfurther separating it from the active processed neurotoxin polypeptide.An antibody which is capable of binding and removing the undesiredpartially processed and unprocessed neurotoxin polypeptide avoids, in anaspect of the invention, interaction with the active processedneurotoxin polypeptide which retains its biological activity. Thanks tothe present invention, purification of neurotoxin is possible wherebythe desired active polypeptide remains essentially unaffected in itsactivity. The skilled worker knows that “activity” is obtained onlyafter proteolytic cleavage of the unprocessed precursor neurotoxinpolypeptide, even though said unprocessed precursor can exert somebiological functions. Accordingly, the “proteolytically processedneurotoxin polypeptide” in an aspect of the invention, is biologicallyactive neurotoxin polypeptide. The term “biologically active” as used inthe present invention relates to the capability of the neurotoxinpolypeptide of subsequent receptor binding, internalization,translocation across the endosomal membrane into the cytosol, and/orendoproteolytic cleavage of one or more proteins involved in synapticvesicle membrane fusion.

It is to be understood that the definitions and explanations of theterms made above apply mutatis mutandis for all aspects described inthis specification in the following except as otherwise indicated.

In another aspect of the present invention, a method for the manufactureof an antibody which specifically binds to unprocessed and/or partiallyprocessed neurotoxin polypeptide is provided, said method comprising thesteps of:

a) contacting a polyclonal antiserum from a non-human animal which hasbeen immunized using a peptide immunogen comprising an amino acidsequence as shown in SEQ ID NO: 25

with a peptide having SEQ ID NO: 25

under conditions which allow for the formation of a complex comprisingthe aforementioned peptide and an antibody which specifically binds tounprocessed or partially processed neurotoxin polypeptide;

b) removing the complex formed in step c) from the antiserum; and

c) releasing the antibody which specifically binds to unprocessed orpartially processed neurotoxin polypeptide from the said complex.

The term “peptide immunogen” as used above refers to an oligopeptidehaving an amino acid sequence as shown in SEQ ID NO: 25 which isprovided in a manner as to allow eliciting of an immune response in anon-human animal. In an aspect said immunogen further comprises KLH andin yet a further aspect, said KLH is linked via a cystein and, in anaspect a C-terminal cystein, to the peptide having SEQ ID NO: 25 via thelinker N-[gamma-maleimidobutyryloxy] succinimide ester (GMBS). How tolink KLH to a peptide by a linker molecule such as GMBS is well known inthe art or described in the accompanying Examples below. In anotheraspect the non-human animal is a mammal, in an aspect a rat, mouse,rabbit, sheep or goat. Prior to carrying out the method of theinvention, a non-human animal which shall be the source of thepolyclonal antiserum will be immunized using the aforementioned peptideimmunogen. How to immunize a non-human animal is well known in the artand described in the accompanying Examples, below. As a result of thesaid immunization, the non-human animal will produce polyclonalantibodies against the peptide immunogen.

A polyclonal antiserum can be obtained from the non-human animal byvarious techniques. In an aspect it is obtained from blood, serum orplasma by standard techniques well known in the art and described in theaccompanying Examples, below. The term “polyclonal antiserum”, thus,includes purified and partially purified sera from the said animal. Sucha polyclonal antiserum is the starting material for the aforementionedmethod. In addition to the desired antibody (or antibodies) whichspecifically binds to unprocessed and/or partially processed neurotoxinpolypeptide, the polyclonal antiserum may comprise additional antibodieswhich do not specifically binds to unprocessed and/or partiallyprocessed neurotoxin polypeptide. These antibodies are separated formthe desired specific antibodies by contacting the polyclonal antiserumwith a peptide also having an amino acid sequence as shown in SEQ ID NO:25. In an aspect, said peptide is immobilized on a carrier as describedin detail elsewhere herein. As a result of the said contacting, acomplex of the peptide and the specific antibodies is formed which cansubsequently be removed from the polyclonal serum. The specificantibodies than can be released from the removed complex. Suitabletechniques for releasing antibodies from such a complex are describedelsewhere herein.

In another aspect said method further comprises prior to step a) thesteps of

i) contacting the said polyclonal antiserum from an non-human animalwhich has been immunized using a peptide immunogen comprising an aminoacid sequence as shown in SEQ ID NO: 25

with the following capture peptides SLD, LDK, and YNK

under conditions which allow for the formation of capture complexescomprising unspecific antibodies comprised by the polyclonal antiserumand the capture peptides; and

ii) removing the capture complexes from the polyclonal antiserum.

In the studies underlying the invention, a polyclonal serum was raisedagainst unprocessed Botulinum neurotoxin type A (BoNT/A), using thelinker peptide coupled to KLH as immunogen (anti-linker peptidescBoNT/A-serum) in goats. Even after affinity purification, the serumshowed cross-reactivity towards processed BoNT/A in a Western blot. Itwas demonstrated that the cross-reactivity depended on the recognitionof tripeptides (SLD, LDK and YNK), which occurred in the linker peptide,as well as, in the light and heavy chains of processed BoNT/A. A secondbatch of the goat immunoserum was purified via two-step affinitychromatography, removing the cross-reactive tripeptide-antibodies. Thesecond anti-linker peptide scBoNT/A-serum displayed no cross-reactivityagainst processed BoNT/A in a western blot. The tripeptides can beapplied, in an aspect, for affinity purification in form of thederivatives shown in any one of SEQ ID Nos. 26 to 28.

In an aspect of the method steps a) to c) are carried out by means ofaffinity chromatography.

Affinity chromatography as used in the present invention refers to atechnique for separating molecules in a mobile phase based on theirdifferent affinities for a stationary phase used in the chromatography.In an aspect, the said technique refers to selective adsorption andsubsequent recovery of a compound from an immobilized ligand. In anotheraspect, the said technique is designed for highly specific and efficientpurification of proteins and related compounds using appropriateselective ligands on beaded and porous matrices for binding targetcompounds, which can then be recovered under mild conditions. The saidtechnique is based on a highly specific interaction such as that betweenantigen and antibody, enzyme and substrate, or receptor and ligand. Inanother aspect said affinity chromatography is perform as columnchromatography. Affinity chromatography as characterized in detail aboveis in one aspect, immunoabsorber chromatography and, hydrophobicinteraction chromatography (HIC), reverse phase chromatography, and inanother aspect, immunoaffinity chromatography applying the binding agentwhich is in even a further aspect, the antibody of the presentinvention. A stationary phase as referred to herein in an aspectconsists of the aforementioned agent as a solid matrix. Said agent is inone aspect, bound to a polypeptide carrier coupled to a solid matrix,and in another aspect, bound to protein A coupled to a solid matrix.

In a further aspect of the aforementioned method steps i) and ii) arecarried out by means of affinity chromatography.

The present invention also pertains to a method for identifying andantibody which specifically binds to unprocessed and/or partiallyprocessed neurotoxin polypeptide comprising the steps of:

a) determining whether the antibody binds to a peptide having an aminoacid sequence as shown in SEQ ID NO: 25; and

b) determining whether the antibody binds to peptides having thefollowing amino acid sequences SLD, LDK and YNK,

wherein an antibody which binds to a peptide having an amino acidsequence as shown in SEQ ID NO: 25 but not to peptides having thefollowing amino acid sequences SLD, LDK and YNK is identified as anantibody which specifically binds to unprocessed and/or partiallyprocessed neurotoxin polypeptide.

The term “determining” as used in accordance with the method foridentifying an antibody encompasses well established techniques fordetermining antibody binding to a given peptide such as immunoblottingtechniques (Western- or Dot-blot technologies), affinity chromatography,plasma surface resonance techniques (BIACORE® Assays) and the like. Itwill be understood that in an aspect the aforementioned binding of theantibody to the peptide or peptides is specific binding (i.e. bindingwithout cross reactivity).

In an aspect, the aforementioned method for identifying an antibody iscarried out for monoclonal antibodies. In an aspect, the method is usedto screen hybridoma cell lines and subsequently produce monoclonalantibodies which specifically bind to unprocessed and/or partiallyprocessed neurotoxin polypeptide. In another aspect, the method can beapplied to screen for polyclonal antibodies, e.g., peptide antibodies,which specifically bind to unprocessed and/or partially processedneurotoxin polypeptide. In an aspect, the method may be applied forconfirmation of the specificity of an antibody manufactured by a methodof the present invention referred to elsewhere in this specification.

The present invention also pertains to an antibody obtainable by theaforementioned method. In aspect the antibody is a polyclonal antibody.In a further aspect said antibody is coupled to a solid support.

The antibody of the invention, in an aspect, allows for the detection ofpartially processed and/or unprocessed neurotoxin polypeptide with ahigh sensitivity and specificity, in an aspect with a limit of detectionof 50 to 80 pg/ml, in an aspect 69 pg/ml.

In principle, the aforementioned antibody can be used for the removal ofpartially processed and/or unprocessed neurotoxin polypeptide fromprocessed neurotoxin polypeptide or for detecting partially processedand/or unprocessed BoNT/A in a sample.

In addition, the present invention relates to a method for manufacturingneurotoxin polypeptide comprising the steps of:

a) contacting a solution containing a mixture of proteolyticallyprocessed, partially processed and/or unprocessed neurotoxinpolypeptides with an agent that specifically binds to unprocessed orpartially processed neurotoxin polypeptides but not to the processedneurotoxin polypeptides under conditions which allow binding of saidagent to the unprocessed or partially processed neurotoxin polypeptideswhereby an agent-complex is formed, andb) removing the agent-complex formed in step a) whereby a solutioncontaining processed neurotoxin polypeptide free of unprocessed orpartially processed neurotoxin polypeptide is obtained.

The term “contacting” as used herein refers to bringing at least twodifferent compounds in physical proximity as to allow physical and/orchemical interaction of said compounds. In accordance with the method ofthis invention, the said two different compounds are, in an aspect, theagent that specifically binds the partially processed or the unprocessedneurotoxin polypeptide which are comprised by the solution. Contactingas meant herein is carried out under conditions and for a time beingsufficient to allow interaction of the agent and the partially processedor the unprocessed neurotoxin polypeptide. Said interaction shall resultin binding of the partially processed or the unprocessed neurotoxinpolypeptide to the agent whereby an antigen-agent complex is formed. Asset forth elsewhere herein, said interaction comprises various kinds ofbinding such as indirect and direct, non-reversible and reversiblemeasures. Suitable conditions which allow for specific interaction ofthe agent and the solution. This is well known to the skilled worker andsaid condition can depend on the agent and the solution to be applied inthe method determined without further ado. Moreover, a time beingsufficient to allow interaction can also be determined by the skilledworker without further ado. Conditions for antibodies as agents aredisclosed in the accompanying examples, below.

A solution as used herein refers to any solvent system containingneurotoxin polypeptide and its partially processed and/or unprocessedneurotoxin polypeptides. The solvent system furthermore comprises asolvent. The solvents encompassed, in various aspects of the invention,are water, aqueous buffer systems, organic solvents, and ionic liquids.In one aspect of the invention, it is an aqueous solvent system.Moreover, the solvent system, in addition to the neurotoxin polypeptideand the solvent may comprise further molecules as well, includingfurther bacterial polypeptides.

The term “agent” as used herein refers to a compound which is capable ofspecifically binding the partially processed or the unprocessedneurotoxin polypeptide. Suitable compounds comprise polypeptides,peptides, antibodies, and organic chemical molecules. In an aspect ofthe present invention, an agent is a polypeptide, peptide or an antibodyas specified elsewhere herein. Said agent in a further aspect of thepresent invention, has at least one binding site for the partiallyprocessed or the unprocessed neurotoxin polypeptide. In another aspectof the invention, said agent has additional binding sites for furtherantibodies which are capable to specifically bind the agent. In evenanother aspect of the invention, the agent is the antibody of thepresent invention as specified above. Moreover, in a further aspect, theagent can comprise different antibodies of the invention. For example,it is conceivable that as an agent in the sense of the invention anantibody according to the invention which specifically binds to thepartially processed neurotoxin polypeptide is used in combination withan antibody of the invention which specifically binds to the unprocessedneurotoxin polypeptide. Alternatively, an agent in the sense of theinvention may comprise two or more different antibodies of the inventionwherein each antibody specifically binds to a different epitope presentin the partially processed and unprocessed neurotoxin polypeptide.

In an aspect of the method of the invention, the agent is immobilized toa matrix as set forth elsewhere herein. In a further aspect, theimmobilization is achieved by covalent direct or indirect binding of theagent to the matrix.

The term “specific binding” as used herein refers to the binding of theagent to the partially processed and/or the unprocessed neurotoxinpolypeptide without any cross reaction with other neurotoxins, host cellproteins, or more other peptides, polypeptides, or other compounds.Specific binding can be tested by various well known techniques. In thisrespect it is referred to the definitions made above in connection withthe antibody of the invention which apply mutatis mutandis.

The term “agent-complex” as used in the present invention refers to theagent bound to the partially processed or to the unprocessed neurotoxinpolypeptide. However, the complex could, in addition, comprise furthermolecules. In an aspect of the invention, the complex can comprisemolecules which stabilize the complex or which facilitate purification,e.g. by allowing interaction of the complex with further molecules orwhich facilitate precipitation of the complex. Additional moleculescomprised by the complex, in an aspect of the invention, encompasssecondary antibodies which specifically bind to the agent or to thecomplex as such. Said secondary antibodies may then also be bound byfurther antibodies or interaction molecules such as polypeptide carriersindirectly or directly. It is to be understood that the complex can alsocomprise further bacterial polypeptides, or other molecules comprised bythe solution.

The term “removing” the antigen-agent complex as used in the presentinvention refers to the separation of the complexed partially processedand of the complexed unprocessed neurotoxin polypeptide from the active,processed neurotoxin containing solution. In one aspect of theinvention, said removing is carried out by means of affinitychromatography, e.g., by using immunobeads, or by immunoprecipitation.

As a consequence of the removal of the partially processed and of theunprocessed neurotoxin polypeptide, the method of the present invention,in an aspect, provides the active processed neurotoxin polypeptide inhighly pure form. The term “highly pure form” as used herein refers, inone aspect, to the active processed neurotoxin polypeptide free ofdetectable amounts of its partially processed or its unprocessedneurotoxin polypeptide, and in another aspect, to active processedneurotoxin polypeptide free of detectable amounts of other impurities aswell. In an aspect, the detectable amount of partially processed orunprocessed neurotoxin is less than 2.5%, less than 1% or, in anotheraspect, less than 0.1%. In a further aspect of the present invention,active processed neurotoxin type A polypeptide as referred to hereinshows under reducing conditions a detectable single band at 100 kDa, anda detectable single band at 50 kDa, but no band at 150 kDa where thepartially processed or the unprocessed neurotoxin type A polypeptidesnormally occur when analyzed, e.g., by SDS-PAGE. It is to be understoodthat other polypeptide impurities can be determined by SDS PAGE as well.It is further to be understood that other serotypes of active processedneurotoxins can be analyzed respectively.

The method of the present invention, wherein said neurotoxin polypeptideis selected from the group consisting of:

a) a neurotoxin polypeptide BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,BoNT/F, BoNT/G or TeNT, and

b) a neurotoxin polypeptide having an amino acid sequence being at least40% identical to the amino acid sequence of the neurotoxin polypeptideof a)

The term “neurotoxin” as used in the present invention refers to theantigenically different serotypes of Botulinum neurotoxins, i.e. BoNT/A,BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, and to TetanusNeurotoxin (TeNT). In an aspect, said BoNT/A has an amino acid sequenceas shown in SEQ ID NO: 17, BoNT/B has an amino acid sequence as shown inSEQ ID NO: 18, BoNT/C1 has an amino acid sequence as shown in SEQ ID NO:19, BoNT/D has an amino acid sequence as shown in SEQ ID NO: 20, BoNT/Ehas an amino acid sequence as shown in SEQ ID NO: 21, BoNT/F has anamino acid sequence as shown in SEQ ID NO: 22, BoNT/G has an amino acidsequence as shown in SEQ ID NO: 23, and TeNT has an amino acid sequenceas shown in SEQ ID NO: 24.

In a further aspect of the method of the present invention, saidneurotoxin polypeptide is a variant of any one of the aforementionedneurotoxin polypeptides which has a sequence which comprises at leastone amino acid substitution, addition and/or deletion with respect toany one of SEQ ID NOs 17 to 24. In another aspect said variantneurotoxin polypeptide has an amino acid sequence being at least 40%sequence identical to the amino acid sequence of BoNT/A (SEQ ID NO: 17),BoNT/B (SEQ ID NO: 18), BoNT/C1 (SEQ ID NO: 19), BoNT/D (SEQ ID NO: 20),BoNT/E (SEQ ID NO: 21), BoNT/F (SEQ ID NO: 22), BoNT/G (SEQ ID NO: 23),or TeNT (SEQ ID NO: 24). In another aspect of the invention, theneurotoxin polypeptide has an amino acid sequence being at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% sequence identical to the amino acid sequenceof BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT. Theterm “identical” as used herein refers to sequence identitycharacterized as determination of the identity of amino acid sequenceswherein the sequences are aligned so that the highest order match isobtained, and which can be calculated using published techniques ormethods codified in computer programs such as, for example, BLASTP,BLASTN, FASTA, Altschul 1990, J Mol Biol 215, 403. The percent identityvalues are in one aspect calculated over the entire amino acid sequence.A series of programs based on a variety of algorithms is available tothe skilled worker for comparing different sequences. In this context,the algorithms of Needleman and Wunsch or Smith and Waterman giveparticularly reliable results. To carry out the sequence alignments, theprogram PileUp (1987, J Mol Evolution 25, 351; Higgins 1989 CABIOS 5,151) or the programs Gap and BestFit (Needleman and Wunsch 1970, J MolBiol 48; 443; Smith and Waterman 1981, Adv Appl Math 2, 482), which arepart of the GCG software packet (Genetics Computer Group 1991, 575Science Drive, Madison, Wis., USA 53711), are to be used. The sequenceidentity values recited above in percent (%) are to be determined, inone aspect of the invention, using the program GAP over the entiresequence region with the following settings: Gap Weight: 50, LengthWeight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which,unless otherwise specified, shall always be used as standard settingsfor sequence alignments.

It will be understood that the aforementioned variants shall, in anaspect of the invention, retain the biological properties ofneurotoxins. Those of skill in the art will appreciate that fullbiological activity is attained only after proteolytic activation, eventhough it is conceivable that the unprocessed precursor can exert somebiological functions or be partially active. “Biological properties” asused herein refers to (a) receptor binding, (b) internalization, (c)translocation across the endosomal membrane into the cytosol, and/or (d)endoproteolytic cleavage of proteins involved in synaptic vesiclemembrane fusion. In vivo assays for assessing biological activityinclude the mouse LD50 assay and the ex vivo mouse hemidiaphragm assayas described by Pearce L B, Borodic G E, First E R, MacCallum RD (1994)(Measurement of botulinum toxin activity: evaluation of the lethalityassay. Toxicol Appl Pharmacol 128: 69-77) and Dressler D, Lange M,Bigalke H (2005) (The mouse diaphragm assay for detection of antibodiesagainst botulinum toxin type B. Mov Disord 20:1617-1619). The biologicalactivity is commonly expressed in Mouse Units (MU). As used herein, 1 MUis the amount of neurotoxic component, which kills 50% of a specifiedmouse population after intraperitoneal injection, i.e. the mouse i.p.LD50 (Schantz & Kauter, 1978). In a further aspect, the variants can beneurotoxins having improved or altered biological properties, e.g., theymay comprise cleavage sites which are improved for enzyme recognition ormay be improved for receptor binding or any other property specifiedabove. It is conceivable that the concept of the present inventionrelies on the presence of one, two or more cleavage sites between lightand heavy chain of the neurotoxin polypeptide while the nature of thecleavage site(s) and the particular amino acid sequence between themdoes not matter as long as the agent is specific for the partiallyprocessed or unprocessed neurotoxin polypeptide. Accordingly, it isanother aspect, to replace protease recognition sites and the linkerpeptide between heavy- and light chain of the neurotoxin polypeptide orflanking sequences surrounding the cleavage site (in case of a singlecleavage site).

In another aspect, the neurotoxin polypeptide in accordance with themethod of the invention may be a chimeric molecule. Such said chimericmolecule, in one aspect, may have single domains substituted.Accordingly, in another aspect, the portion of the neurotoxin heavychain is replaced by a portion of an FC domain of an antibody.

In an aspect, the neurotoxin polypeptide produced according to themethod of the present invention may be used for analytical toolsincluding ELISA, antigens for ELISA, and control standards.

To achieve a neurotoxin preparation being free of other impurities aswell, further steps of purification well known in the art can be addedto the aforementioned method of the present invention and will beexplained in the following.

As follows from the above in one aspect of the method of the presentinvention, said method is performed by means of affinity chromatography.

-   -   In another aspect of the invention, the specific immunoabsorber        is prepared for the immunoaffinity chromatography as follows:    -   synthesis of the specific oligopeptide (represented by any one        of the SEQ ID NOs: 1 to 16 or 25) of the unprocessed or the        partially processed precursor polypeptide in particular,        preparation of a synthetic oligopeptide;    -   conjugation of the peptide to a suitable carrier for        immunization (including hemocyanin, BSA, lipopolysaccarides, and        other) specifically, binding of the oligopeptide to a        polypeptide carrier;    -   immunization of animals to produce poly- or monoclonal        antibodies in particular, immunization of rabbits or goats to        produce polyclonal, and immunization of mice to produce        monoclonal antibodies (at least ten animals need to be        immunized, in order to obtain an affine antibody;    -   hybridoma cell lines are generated to produce monoclonal        antibodies;    -   purification of the antibodies by conventional and affinity        chromatography (for the latter the oligopeptide will be bound to        a carrier) specifically, the antibodies are purified using for        example Protein A or G and/or via the oligopeptide bound to a        carrier (the latter was used for immunization) or via peptide        affinity chromatography for removing unspecific antibodies        followed by affinity chromatography;    -   cleavage of the specific antibodies in Fab fragments in        particular, the specific antibodies are treated with a protease        such as Papain in order to obtain the respective Fab fragments;    -   the Fab fragments are characterized to their binding properties        prior to further applications;    -   the antibodies will be coupled to a column matrix such as        activated sepharose in particular, specific Fab fragments are        coupled to an active linkage group of a carrier material;    -   the immuno-absorber (in a column) is washed and equilibrated        using a suitable buffer system;    -   the unprocessed or the partially processed precursor neurotoxin        polypeptide is specifically bound to the immunoabsorber whereas        the active, processed neurotoxin polypeptide passes through the        column unchanged (without being bound to) and will be collected;

In another aspect of the method of the invention, size exclusionchromatography is performed in addition. By size exclusionchromatography as used in the present invention, particles are separatedbased on their size, i.e. on their hydrodynamic volume. A mobile phaseis either an aqueous solution used to transport the sample (gelfiltration chromatography), or an organic solvent (gel permeationchromatography). A stationary phase is either a gel medium(polyacrylamide, dextran or agarose) and filter under low pressure, or asilica, or crosslinked polystyrene medium under a higher pressure. Ineven another aspect, said size exclusion chromatography is performed ascolumn chromatography. In a further aspect of the method of the presentinvention, said size exclusion chromatography is performed usingmolecular sieves with distinct pore sizes such as activated carbon,silica gel, zeolite.

The method of the present invention, in another aspect, furthercomprises ion exchange chromatography.

Ion exchange chromatography as used in the present invention separatesmolecules based on differences between the overall charge of theproteins and related compounds. It is used for protein purification, forpurification of oligonucleotides, peptides, or other charged molecules.Such molecules may be present in the solution to be applied to themethod of the purification as contaminations. The protein or the relatedcompound of interest, in the present case the Neurotoxin, must have acharge opposite to that of the functional group attached to the resin inorder to bind. Because this interaction is ionic, binding must takeplace under low ionic conditions. Elution is achieved by increasing theionic strength to break up the ionic interaction, or by changing the pHof the protein. In an aspect of the method of the invention, saidexchange chromatography is performed as column chromatography.

In one aspect, exchange chromatography as used in accordance with thepresent invention is ion exchange chromatography.

The ion exchange chromatography as used in the present invention is in afurther aspect performed by cation and/or anion chromatography. In anionexchange chromatography as used herein the surface charge of the solutes(proteins, peptides, nucleic acids, endotoxins) which bind will be netnegative, thus to get binding of a specific protein one should be nearor above the pI of that protein. Commonly used anion exchange resins areQ-resin (Q Sepharose), a Quaternary amine; and DEAE (DiEthylAminoEthane)resin. Generally, an ion exchange resin is an insoluble matrix of smallbeads having a charged surface, used as an artificial zeolite. Differenttypes of resins can be distinguished based on their functional groupsincluding strongly acidic resins (sulfonic acid groups, eg. sodiumpolystyrene sulfonate or polyAMPS), strongly basic resins, (quaternaryamino groups, e.g. trimethylammonium groups, eg. polyAPTAC), weaklyacidic resins (mostly, carboxylic acid groups), weakly basic resins(primary, secondary, and/or ternary amino groups, e.g., polyethyleneamine). There are also specialised types of resins can be furtherdistinguishes including chelating resins (iminodiacetic acid, thiourea).

In cation exchange chromatography as used herein, the surface charge ofthe solutes (proteins, peptides, nucleic acids, endotoxins) which bindwill be net positive, thus to get binding of a specific protein oneshould be near or below the pI of that protein. Commonly used cationexchange resins are S-resin, sulfate derivatives; and CM resins,carboxylate derived ions.

In an aspect of the method of the present invention said ion exchangechromatography is carried out prior to and/or after affinitychromatography. In another aspect of the method of the invention, saidion exchange chromatography as used herein is carried out prior to theaffinity chromatography of the present invention.

Due to this measure, the risk of potential cross-reactivity orunspecific binding during affinity chromatography can be further avoidedand reduced.

The method of the present invention allows for the manufacture of activeprocessed neurotoxin free of unprocessed or partially processedprecursor polypeptide and thus, obtaining higher amounts of the activeprocessed neurotoxin polypeptide.

The present invention refers, in principle, to the use of the antibodyof the present invention for separating the active processed neurotoxinfrom its unprocessed or partially processed precursor polypeptide. Inone aspect, the antibody of the present invention is used for theseparation of the unprocessed or partially processed precursorneurotoxin polypeptide from the active processed neurotoxin polypeptide,in solution containing a mixture of said polypeptides, and, thus,obtaining active processed neurotoxin polypeptide free of an unprocessedor partially processed precursor neurotoxin polypeptide as described indetail elsewhere herein.

The present invention also relates to a method for the manufacture of amedicament comprising the steps of the aforementioned method and thefurther step of formulating the proteolytically processed neurotoxinpolypeptide as medicament.

The term “medicament” as used herein refers, in one aspect, to apharmaceutical composition containing the biologically active(proteolytically processed) neurotoxin polypeptide as pharmaceuticalactive compound, wherein the pharmaceutical composition may be used forhuman or non-human therapy of various diseases or disorders in atherapeutically effective dose.

A pharmaceutical composition as used herein comprises the biologicallyactive (proteolytically processed) Neurotoxin polypeptide of the presentinvention, and in one aspect, one or more pharmaceutically acceptablecarrier. The active Neurotoxin can be present in liquid or lyophilizedform. In an aspect, said compound can be present together with glycerol,protein stabilizers (e.g., human serum albumin (HAS)) or non-proteinstabilizers.

The pharmaceutical composition is, in one aspect, administeredtopically. Conventionally used drug administration is administeredintra-muscular, subcutaneous (near glands). However, depending on thenature and the mode of action of a compound the pharmaceuticalcomposition may be administered by other routes as well.

The compound, i.e. the biologically active (proteolytically processed)neurotoxin polypeptide is the active ingredient of the composition, andis in one aspect administered in conventional dosage forms prepared bycombining the drug with standard pharmaceutical carriers according toconventional procedures. These procedures may involve mixing,granulating, and compression, or dissolving the ingredients asappropriate to the desired preparation. It will be appreciated that theform and character of the pharmaceutical acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration, and other well-known variables.

The carrier(s) must be acceptable in the sense of being compatible withthe other ingredients of the formulation and being not deleterious tothe recipient thereof. The pharmaceutical carrier employed may include asolid, a gel, or a liquid. Exemplary of solid carriers are lactose,terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesiumstearate, stearic acid and the like. Exemplary of liquid carriers arephosphate buffered saline solution, syrup, oil, water, emulsions,various types of wetting agents, and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax. Saidsuitable carriers comprise those mentioned above and others well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa.

The diluent(s) is/are selected so as not to affect the biologicalactivity of the combination. Examples of such diluents are distilledwater, physiological saline, Ringer's solutions, dextrose solution, andHank's solution. In addition, the pharmaceutical composition orformulation may also include other carriers, adjuvants, or non-toxic,non-therapeutic, non-immunogenic stabilizers and the like.

A therapeutically effective dose refers to an amount of the compound tobe used in a pharmaceutical composition of the present invention whichprevents, ameliorates or treats the symptoms accompanying a disease orcondition referred to in this specification. Therapeutic efficacy andtoxicity of the compound can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population) and LD50 (thedose lethal to 50% of the population). The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50.

The dosage regimen will be determined by the attending physician andother clinical factors. As is well known in the medical arts, dosagesfor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Progress can be monitoredby periodic assessment.

The pharmaceutical compositions and formulations referred to herein areadministered at least once in order to treat or ameliorate or prevent adisease or condition recited in this specification. However, the saidpharmaceutical compositions may be administered more than one time.

Specific pharmaceutical compositions are prepared in a manner well knownin the pharmaceutical art and comprise at least one active compoundreferred to herein above in admixture or otherwise associated with apharmaceutically acceptable carrier or diluent. For making thosespecific pharmaceutical compositions, the active compound(s) willusually be mixed with a carrier or the diluent. The resultingformulations are to be adapted to the mode of administration. Dosagerecommendations shall be indicated in the prescribers or usersinstructions in order to anticipate dose adjustments depending on theconsidered recipient.

The medicament according to the present invention may in a furtheraspect of the invention comprise drugs in addition to the biologicallyactive (proteolytically processed) neurotoxin polypeptide which areadded to the pharmaceutical composition during its formulation. Finally,it is to be understood that the formulation of a pharmaceuticalcomposition takes place under GMP standardized conditions or the like inorder to ensure quality, pharmaceutical security, and effectiveness ofthe medicament.

The present invention, in general, contemplates a composition comprisingproteolytically processed neurotoxin polypeptide obtainable by themethod of the present invention.

The term “composition” refers to any composition formulated in solid,liquid, aerosol (or gaseous) form. Said composition comprises thecompound of the invention optionally together with suitable auxiliarycompounds such as diluents or carriers or further ingredients. In thiscontext, it is distinguished for the present invention between auxiliarycompounds, i.e. compounds which do not contribute to the effectselicited by the compound of the present invention upon application ofthe composition for its desired purpose, and further ingredients, i.e.compounds which contribute a further effect or modulate the effect ofthe compound of the present invention. Suitable diluents and/or carriersdepend on the purpose for which the composition is to be used and theother ingredients. The person skilled in the art can determine suchsuitable diluents and/or carriers without further ado. Examples ofsuitable carriers and/or diluents are disclosed elsewhere herein.

In a further aspect of the invention, the aforementioned composition isa medicament as specified elsewhere in the description in more detail.In one aspect the said medicament can be used for prevention and/ortreatment of at least one of the following diseases and disorders:voluntary muscle strength, focal dystonia, including cervical, cranialdystonia, and benign essential blepharospasm, hemifacial spasm, andfocal spasticity, gastrointestinal disorders, hyperhidrosis, andcosmetic wrinkle correction, in a further aspect also Blepharospasm,oromandibular dystonia, jaw opening type, jaw closing type, bruxism,Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervicaldystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngealdystonia, laryngeal dystonia, spasmodic dysphonia/adductor type,spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, armdystonia, task specific dystonia, writer's cramp, musician's cramps,golfer's cramp, leg dystonia, thigh adduction, thigh abduction kneeflexion, knee extension, ankle flexion, ankle extension, equinovarus,deformity foot dystonia, striatal toe, toe flexion, toe extension, axialdystonia, pisa syndrome, belly dancer dystonia, segmental dystonia,hemidystonia, generalised dystonia, dystonia in lubag, dystonia incorticobasal degeneration, dystonia in lubag, tardive dystonia, dystoniain spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia inHuntington's disease, dystonia in Hallervorden Spatz disease,dopa-induced dyskinesias/dopa-induced dystonia, tardivedyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias,kinesiogenic non-kinesiogenic action-induced palatal myoclonus,myoclonus myokymia, rigidity, benign muscle cramps, hereditary chintrembling, paradoxic jaw muscle activity, hemimasticatory spasms,hypertrophic branchial myopathy, maseteric hypertrophy, tibialisanterior hypertrophy, nystagmus, oscillopsia supranuclear gaze palsy,epilepsia, partialis continua, planning of spasmodic torticollisoperation, abductor vocal cord paralysis, recalcitant mutationaldysphonia, upper oesophageal sphincter dysfunction, vocal foldgranuloma, stuttering Gilles de Ia Tourette syndrome, middle earmyoclonus, protective larynx closure, postlaryngectomy, speech failure,protective ptosis, entropion sphincter Odii dysfunction,pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus,postoperative immobilisation tremor, bladder dysfunction, detrusorsphincter dyssynergia, bladder sphincter spasm, hemifacial spasm,reinnervation dyskinesias, cosmetic use craw's feet, frowning facialasymmetries, mentalis dimples, stiff person syndrome, tetanus prostatehyperplasia, adipositas, treatment infantile cerebral palsy strabismus,mixed paralytic concomitant, after retinal detachment surgery, aftercataract surgery, in aphakia myositic strabismus, myopathic strabismus,dissociated vertical deviation, as an adjunct to strabismus surgery,esotropia, exotropia, achalasia, anal fissures, exocrine glandhyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis,axillar palmar plantar rhinorrhea, relative hypersalivation in stroke,in Parkinsosn's, in amyotrophic lateral sclerosis spastic conditions, inencephalitis and myelitis autoimmune processes, multiple sclerosis,transverse myelitis, Devic syndrome, viral infections, bacterialinfections, parasitic infections, fungal infections, in hereditaryspastic paraparesis postapoplectic syndrome hemispheric infarction,brainstem infarction, myelon infarction, in central nervous systemtrauma, hemispheric lesions, brainstem lesions, myelon lesion, incentral nervous system hemorrhage, intracerebral hemorrhage,subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage,in neoplasias, hemispheric tumors, brainstem tumors, myelon tumors. Fordetails and symptoms see, e.g., Jost 2007, Drugs 67(5), 669 or Dressier2000 in Botulinum Toxin Therapy, Thieme Verlag, Stuttgart, N.Y.

In another aspect of the invention, the composition is a cosmeticcomposition which can be formulated as described for a pharmaceuticalcomposition above. For a cosmetic composition, likewise, it is envisagedthat the compound of the present invention is in an aspect used insubstantially pure form. Cosmetic compositions are, in a further aspect,to be applied intramuscular. In an even further aspect of the invention,cosmetic compositions comprising the neurotoxin can be formulated as ananti-wrinkle solution.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

The Figures Show:

FIG. 1: Scheme of the conventional chromatographic purification ofneurotoxin polypeptide.

FIG. 2: Scheme of the chromatographic purification of biologicallyactive (proteolytically processed) neurotoxin polypeptide and theseparation of its partially processed or unprocessed polypeptideprecursor according to the present invention.

FIG. 3: Western blot using an antibody which specifically recognizes SEQID NO: 25 and which has been obtained by the method of the presentinvention. Size of the bands is indicated in kDa. The individual lanesare explained in the Examples.

The following Examples illustrate the invention and shall, whatsoever,not be construed to limit its scope.

EXAMPLES Example 1 Generation of Immunogen and Antibodies

Generation of Immunogens

1. Linkerpeptid-Immunogen I: The peptide with the sequenceNH₂-TKSLDKGYNK-C-COOH was generated by an external provider and thencoupled by the linker GMBS to the carrier-protein KLH.

2. Linkerpeptid-Immunogen II: a) Activation of ovalbumin; 2.18 mgsulfo-smcc(sulfosuccinimidyl-4(N-maleimidomethyl)cyclohexane-1-carboxylate) weresolved in 50 μl DMSO. Subsequently, 2.5 ml ovalbumin solution containing7.5 mg/ml ovalbumin (buffer: 5 mM sodiumphosphate; 0.9% NaCl) were addedand the solution was incubated for 1 h at room temperature withrotation. A buffer change was performed using PD10 columns, activatedovalbumin was eluted in 3.5 ml buffer containing 10 mM sodiumphosphate;0.9% NaCl. b) Coupling of the peptide to ovalbumin; 8 mg of the peptidAc-DKGYNC-OH were solved in 250 μl H₂O and 2.5 μl 500 mM TCEP HCL(tris[2-carboxethyl]phosphine HCL) and subsequently neutralized with 1mM NaoH. Finally, activated ovalbumin was added and the reaction mixturewas incubated at room temperature for 4.4 h with rotation. By adding a10 mM cysteine solution remaining reactive residues were blocked byincubation for 1 h with rotation. A dialysis was performed using 10 mMsodiumphosphate; 0.9% NaCl.

Immunization

Antisera were obtained by immunization.

1.) Anti-linkerpeptide scBoNT/A-serum I: As immunogen the linkerpeptideimmunogen I was used which was coupled by the linker GMBS to thecarrier-protein KLH. Two goats were immunized subcutaneously each firstwith 300 μg dekapeptid immunogen in Freud'schem adjuvant and finallyimmunized for four times in a 2 week rhythm with 100 μg immunogen inincomplete freud' schem adjuvant. After 49, 63, 77 and 84 days antiserawere collected. Affinity chromatography was performed using the serumcollected from the last bleeding on day 84.

2.) Anti-linkerpeptide scBoNT/A-serum II: As immunogen the linkerpeptideimmunogen II was used which was coupled by the linker SMCC to thecarrier-protein ovalbumin. Two rabbits were immunized intradermal eachfirst with 300 μg linkerpeptide immunogen II in freud' schem adjuvantand finally immunized for five times in a 2 week rhythm with 150 μglinkerpeptide immunogen II in Montanide ISA 206. Affinity chromatographywas performed using the serum collected from the bleeding on day 60 or110, respectively.

Two Step Affinity Chromatography of the Sera

1. Generation of the matrix: For the two step affinity chromatographytwo different ultra link iodoacetyl matrices containing differentpeptides were generated.

On the one hand site the cross reactive peptides SLD, LDK and YNK werepresented in form of the following peptides Ac-ELDKYN-C-COOH (SEQ ID NO:26), NH₂-NISLDL-C-COOH (SEEQ ID NO: 27) and NH₂-YYNKF-C-COOH (SEQ ID NO:28) and were coupled to the matrix using the general description givenbelow. On the other hand the linker peptide (SEQ ID NO: 25) was coupledto the matrix using the general description given below in the form ofthe following derivative: Ac-TKSLDKGYNKA-C-COOH.

General Description:

Coupling Buffer: 50 mM Tris, 5 mM EDTA-Na, pH 8.5. Prepare a volume ofbuffer equal to 20 times the volume of UltraLink® Iodoacyl Gel to beused.

L-Cysteine HCL; Wash solution: 1 mM sodium chloride (NaCl).

Empty gravity-flow or spin column that may be capped both top andbottom:

Prepare the Peptide or Protein Sample

Dissolve the peptide with Coupling Buffer.

Couple to UltraLink® Iodoacyl Gel:

1. With the bottom cap in place on a gravity-flow column, add thedesired quantity of the UltraLink® Iodoacyl Gel slurry, allow the gel tosettle for 15 minutes.

2. Drain the liquid from the packed column and wash/equilibrate theUltraLink® Iodoacyl Gel with 5 gel-bed volumes of Coupling Buffer byadding buffer to the top of the gel bed allowing to drain through thecolumn. Do not allow the gel bed to run dry.

3. Replace bottom cap and add the prepared sulfhydryl-containing sample.Approximately 1 ml of sample solution can be applied per ml ofUltraLink® Iodoacyl Gel.

4. Replace the top cap and mix column at RT for 15 minutes.

5. Stand the column upright and incubate at RT for 30 minutes withoutmixing.

6. Sequentially remove top and bottom column caps and allow the solutionto drain.

7. Wash column with three gel-bed volumes of Coupling Buffer.

Block Nonspecific Binding Sites on Gel.

1. Replace the bottom cap on column.

2. Prepare a solution of 50 mM L-Cysteine HCL in Coupling Buffer and add1 ml of this solution to the column for each milliliter of gel.

3. Replace the top cap and mix for 15 minutes at RT, then incubate thereaction without mixing for an additional 30 minutes at RT.

2. Two step affinity chromatography:

Sera to be purified are first separated from blood.

The crude serum is given on the first column containing the crossreactive tripeptides. The cross reactive antibodies bind to thetripeptides and are separated from the crude serum. The filtrate of thisfirst column is given to the second column containing the boundlinkerpeptide. The linkerpeptide specific antibodies bind to thelinkerpeptide. Low affinity anti-linkerpeptide scBoNT/A antibodies areremoved from the column by a high stringency wash with PBS buffer (0.5 MNaCl). Subsequently, the bound high affinity anti linkerpeptide scBoNT/Aantibodies are eluted and concentrated. This concentrate corresponds tothe used anti linkerpeptide scBoNT/A serum.

Example 2 Test and Verification of Antibody Specificity

Reagents ELISA:

Coating buffer: 0.005 M-1M Tris; 0.9% NaCl, preferable 0.01 M-0.2 MTris; 0.9% NaCl, pH=8.5.

Catcher antibody: anti linkerpeptide scBoNT/A serum.

Blocking and antibody diluent buffer: 0.5-5% BSA in 0.01 M sodiumphosphate; 0.9% NaCl, pH=7.4.

Sample buffer: 0.5%-5% BSA in 0.005 M-1 M sodium phosphate; 0.1-0.5 MNaCl; 0.01%-1 Tween 20, preferably 1%-3% BSA in 0.005-0.1 M sodiumphosphate; 0.15 M-0.4 M NaCl; 0.05%-0.5% Tween 20, pH=7.4.

Wash buffer: 0.01 M sodium phosphate; 0.9% NaCl; 0.05% Tween 20, pH=7.4.

Detection antibody: monoclonal antibody against BoNT/A.

Secondary antibody: A polyclonal anti mouse IgG (H&L) antibodyconjugated to peroxidase.

Substrate: TMB, commercially available.

2. Reagents Western Blot:

Denaturating sample buffer, commercially available.

SDS gel, commercially available.

MES running buffer (SDS PAGE): commercially available.

PVDF membrane: commercially available.

Transfer buffer (Western Blot): commercially available.

Sample: Botulinum Neurotoxin A with Dichain-BoNT/A and scBoNT/A.

Primary antibody: anti linkerpeptid scBoNT/A serum.

Secondary antibody: polyclonal donkey anti goat antibody IgG (H&L)conjugated to alkaline phosphatase.

Blocking and antibody diluent buffer: 0.5%-5% BSA in 0.01 M-0.1 M Tris;0.9% NaCl; 0.05-5% Tween 20, pH=7.4.

Washing buffer: 0.01 M-0.1 M Tris; 0.9% NaCl; 0.05-5 Tween 20, pH=7.4.

Tris buffer: 0.025 M Tris, pH=8.0.

Substrate: BCIP/NBT, commercially available.

a) Specificity of the antiserum with regard to BoNT/B and BoNT/E: Fordetermining the specificity of the antisera with regard to BoNT/B andBoNT/E the recovery rate of substances were analyzed in ELISA.Microtiter plates are incubated with 100 μl/well of coating buffercontaining 0.5 μg anti linkerpeptide scBoNT/A-serum/ml for 16 h at roomtemperature and subsequently washed three times with washing buffer. 200μl/well blocking solution is added to the microtiter plates andincubated for 1 h at room temperature. The antigen scBoNT/A (dilutionseries in sample buffer; pg/ml concentration) is used as a calibrationstandard, microtiter plates are incubated with 100 μl/well calibrationstandard. BoNT/B or BoNT/E, respectively are diluted in sample bufferand applied to the microtiter plate in a volume of 100 μl/well. Bothsubstances are applied in excess, a dilution of 200 ng/ml is used.Samples and standards are incubated for 2 h at 37° C. Microtiter platesare washed three times with washing buffer. 100 μl of detectionbuffer/well are added and incubated for 1 h at room temperature. Thenmicrotiter plates are washed three times with washing buffer.Subsequently, the incubation with 100 μl/well of the secondary antibodyfor 1 h at room temperature is performed. Then microtiter plates arewashed three times with washing buffer.

The detection reaction is started by adding 100 μl substrate/well. Afterincubation for 30 minutes at room temperature the reaction is stopped byadding 50 μl 2 M H₂SO₄/well and the absorbance is determined at 450 nm.For determination of specificity the concentrations of BoNT/b and BoNT/Eare calculated by standardization. By calculating the recovery rate thespecificity of the anti linkerpeptides scBoNT/A for sterotypes B and Ecan be determined. The lower the recovery rate, the lower the crossreactivity and the better the specificity of the serum in regard toscBoNT/A.

b) Specificity of the anti-linkerpeptide scBoNT/A with regard to DichainBoNT/A: For determination of specificity of the antiserum in regard toactivated Dichain-BoNT/A an immunohistological detection by Westernblotting is performed. A NT sample (scBoNT/A at least 50 ng,Dichain-BoNT/A dependent on the sample used) is separated under reducingconditions by SDS-PAGE in accordance to their molecular weight intoscBoNT/A, LC and HC (Dichain-BoNT/A). The proteins are then blotted ontoa PVDF membrane. The membrane is blocked with 20 ml blocking buffer for1 h at room temperature. The blocking buffer is removed and 20 ml ofprimary antibody solution containing 0.005 μg/ml anti linkerpeptidescBoNT/A serum are added. The primary antibody is incubated over nightat 4° C. The antibody containing solution is removed and the membrane iswashed three times for 30 minutes with 20 ml washing buffer at 37° C.Subsequently, the membrane is incubated for 3 h at room temperature with20 ml of the secondary antibody in a concentration of 0.4 μg/ml. Thesecondary antibody solution is removed and the membrane is washed threetimes for 30 minutes with 20 ml washing buffer at 37° C. Additionally,the membrane is washed once with 20 ml of a 25 mM TRIS buffer for 5minutes at room temperature.

The detection reaction is performed by adding the substrate. Thesubstrate is incubated for 15 minutes and the color reaction is stoppedby adding water. The specificity is determined by the staining of thescBoNT/A band at 150 kDa. Specificity of the anti linkerpeptide wasdetermined when only the 150 kDa specific band was detected but no bandspecific for Dichain BoNT/A at 100 kDa (HC) and 50 kDa (LC). Fig: 3shows in lane 3 the specificity for the 150 kDa scBoNT/A of a BoNT/Apreparation (NT sample, see above). No bands are apparent at 100 kDa or50 kDa, only the scBoNT/A is recognized. For comparison, in lane 4, ablend of partially processed and unprocessed scBoNT/A is shown and lane5 shows a non-cleavable scBoNT/A control. Buffer control is shown inlane 2.

The invention claimed is:
 1. A pharmaceutical composition comprisingproteolytically processed, biologically active dichain neurotoxin,wherein the composition is free of detectable amounts of partiallyprocessed or unprocessed neurotoxin polypeptides, wherein theproteolytically processed, biologically active dichain neurotoxin isobtained by the steps of: a) contacting a solution comprising a mixtureof processed and partially processed and/or unprocessed neurotoxinpolypeptides with an antibody which has been raised against a peptideimmunogen comprising the amino acid sequence of SEQ ID NO: 25; b)allowing the binding of the antibody to the unprocessed and/or partiallyprocessed neurotoxin polypeptides whereby complexes comprising theantibody and partially processed or unprocessed neurotoxin polypeptidesare formed; and c) removing the complexes formed in step b) to obtain asolution of processed, active dichain neurotoxin, wherein the solutionis free of detectable amounts of unprocessed and/or partially processedneurotoxin polypeptides; and d) formulating the solution of processedneurotoxin polypeptides obtained in step c) with at least one stabilizerof the biologically active dichain neurotoxin selected from proteinstabilizers and non-protein stabilizers in pharmaceutically acceptablediluents, carriers, and/or adjuvants.
 2. A method for obtaining apurified solution of processed neurotoxin polypeptides comprising thesteps of: a) contacting a solution comprising a mixture of processed andpartially processed and/or unprocessed neurotoxin polypeptides with anantibody which has been raised against a peptide immunogen comprisingthe amino acid sequence of SEQ ID NO: 25; b) allowing the binding of theantibody to the unprocessed and/or partially processed neurotoxinpolypeptides whereby complexes comprising the antibody and partiallyprocessed and/or unprocessed neurotoxin polypeptides are formed; c)removing the complexes formed in step b) to obtain a solution ofprocessed neurotoxin polypeptides which is flee of unprocessed and/orpartially processed neurotoxin polypeptides; and d) formulating thesolution of processed neurotoxin polypeptides obtained in step c) withat least one stabilizer of the biologically active dichain neurotoxinselected from protein stabilizers and non-protein stabilizers.
 3. Themethod of claim 2, wherein steps a) to c) are carried out by affinitychromatography.
 4. The method of claim 2, further comprising an onexchange chromatography step.
 5. The method of claim 2, furthercomprising formulating the solution of processed neurotoxin polypeptidesobtained in step d) in pharmaceutically acceptable diluents, carriersand/or adjuvants.